Oxidation of hydrocarbons



United States Patent 3,057,784 OXIDATION OF HYDROCARBONS John B. Davis and Richard L. Raymond, Dallas, Tex., assignors to Socony Mobil Oil Company, Inc., a corporation of New York No Drawing. Filed Nov. 2, 1959, Ser. No. 850,015 8 Claims. (Cl. 195-28) This invention relates to oxidation of hydrocarbons and relates more particularly to oxidation by microbiological means of alkyl substituted cyclic hydrocarbons.

It has heretofore been proposed to oxidize cyclic hydrocarbons containing an alkyl substituent by microbiological means. Where the alkyl substituent contains a long carbon chain, the oxidative reaction goes to completion. However, with this type of hydrocarbon, oxidative deterioration, i.e., conversion to carbon dioxide and water, tends to be the predominant reaction, and little, if any, of the desired oxidized hydrocarbon product is obtained. On the other hand, where the alkyl substituent contains only a short carbon chain, maintenance of the oxidation reaction cannot be effected. Upon admixture of the hydrocarbon with the culture containing the oxidative microorganism, the oxidation reaction begins. But, shortly thereafter, the oxidation reaction ceases even though a considerable portion of the hydrocarbon remains in the mixture. Thus, only a small amount of any of the desired oxidized hydrocarbon product is obtained.

It is an object of this invention to provide a method for microbiologically oxidizing an alkyl substituted cyclic hydrocarbon. It is another object of this invention to minimize oxidative deterioration of the hydrocarbon in microbiological oxidation of an alkyl substituted cyclic hydrocarbon containing a long-chain alkyl substituent. It is another object of this invention to increase the yield of desired oxidized hydrocarbon product from microbiological oxidation of an alkyl substituted cyclic hydrocarbon. It is another object of this invention to provide a means for carrying to completion the microbiological oxidation of alkyl substituted cyclic hydrocarbons containing only short-chain alkyl substituents. Other objects of this invention will become apparent from the following detailed description.

In accordance with the invention, the microbiological oxidation of an alkyl substituted cyclic hydrocarbon is carried out in a fermentation reaction mixture containing in addition to the alkyl substituted cyclic hydrocarbon a substrate capable of supporting the growth of the oxidative microorganism.

While we do not wish to limit our invention to the con sequences of any theory, we believe, on the basis of the evidence provided by our process, that a cyclic hydrocarbon containing only a short-chain alkyl substituent does not support the growth of the oxidative microorganism. Apparently, the oxidative microorganism is capable of utilizing for growth an alkyl substituent on a cyclic hydrocarbon. However, this alkyl substituent must contain a minimum number of carbon atoms. It is known that, where the alkyl substituent contains, for example, at least nine carbon atoms, the fermentation reaction will continue to completion. Thus, it would appear that the oxidative microorganism requires an alkyl substituent containing at least nine carbon atoms for growth. But, since with a cyclic hydrocarbon containing a long-chain alkyl substituent, oxidative deterioration occurs, it would also appear that growth at the expense of the alkyl substituent involves oxidative deterioration. Toward the other end of the scale, however, where the alkyl substituent contains three carbon atoms, for example, the fermentation reaction will not continue to completion. The conclusion is that growth of the oxidative microorganism is not Patented Oct. 9, 1962 supported by the substituent containing three carbon atoms. 0n the other hand, by providing in the fermentation reaction mixture, in accordance with our process, in addition to the alkyl substituted cyclic hydrocarbon to be oxidized, a substrate capable of supporting the growth of the oxidative microorganism, the oxidation reaction continus until the reaction mixture no longer contains unreacted alkyl substituted cyclic hydrocarbon even where the alkyl substituent contains only three carbon atoms. The reaction product is a desired oxidized hydrocarbon. Oxidative deterioration is not the predominant reaction and the desired oxidized hydrocarbon is obtained in high yield. Thus, we believe the oxidative microorganism grows at the expense of the added substrate but utilizes the alkyl substituent of the cyclic hydrocarbon as a source of energy with consequent oxidation of the cyclic hydro carbon.

Various alkyl substituted cyclic hydrocarbons can be oxidized by the process of the invention. Included among these cyclic hydrocarbons are the aromatic hydrocarbons and the naphthenes. Thus, the process of theinvention can be employed for the oxidation of the alkyl substituted benzenes and the alkyl substituted 5- and 6-membered cyclo parafl'lns. The cyclic hydrocarbons can contain a single ring but may also contain more than one ring.

One, or two or more, alkyl substituents can be on the ring portion of the cyclic hydrocarbon. Any number of carbon atoms may be in the alkyl substituent. Thus, the alkyl substituents can be those containing one, two, three, or more, carbon atoms. The alkyl substituent can be a straight-chain 'or a branched-chain substituent. Where one or more alkyl substituents are present one may be a branched-chain substituent and the other may be a straight-chain substituent.

Alkyl substituted aromatic cyclic hydrocarbons which may be oxidized by the process of the invention include methylbenzene, or toluene, the dimethylbenzenes or xylenes, the trimethylbenzenes such as mesitylene, ethylbenzene, the diethylbenzenes, the isomeric propylbenzenes such as cumene, the isomeric butylbenzenes, the isomeric amylbenzenes, the isomeric hexylbenzenes, the isomeric heptylbenzenes, and the isomeric octylbenzenes. Other alkyl substituted aromatic hydrocarbons which may be employed include p-cymene, methylnaphthalene, and ethylnaphthalene.

Included among the alkyl substituted naphthenes which may be treated by the process of the invention are methylcyclopentane, the dimethylcyclopentanes, the trimethylcyclopentanes, ethylcyclopentane, the diethylcyclopentanes, the isomeric propylcyclopentanes, the isomeric butylcyclopentanes, the isomeric amylcyclopentanes, the isomeric hexylcyclopentanes, the isomeric heptylcyclopentanes, and the isomeric octylcyclopentanes. Also included among these naphthenes are methylcyclohexane, the dimethylcyclohexanes, the trimethylcyclohexanes, the tetramethylcyclohexanes, ethylcyclohexane, the isomeric propylcyclohexanes, isopropyl-4-methylcyclohexane, the isomeric butylcyclohexanes, the isomeric amylcyclohexanes, the isomeric hexylcyclohexanes, the isomeric heptylcyclohexanes, and the isomeric octylcyclohexanes.

Microbiological oxidation of the alkyl substituted cyclic hydrocarbons proceeds along the route of oxidation of the alkyl substituent. With normal alkyl substituents, this oxidation is beta oxidation, namely, oxidation of the terminal carbon atom followed by oxidation of each alternate carbon atom. Various alkyl substituents are more susceptible to microbiological oxidation than others. Thus, where there are two alkyl substituents on the cyclic group, one may be oxidized to the exclusion of the other. For example, with p-cymene, the methyl group will be oxidized while the isopropyl group will not be afiected.

Ethylbenzene n-Propylbenzene n-Butylbenzene n-Dodecylbenzene p-Cymene n-Butylcyclohexane n-Nonylbenzene Phenylacetic acid Bcnzoic acid Phenylacetic acid Phenylacetic acid p-Isopropylbenzoic acid Cyclohexaneacetic acid Benzoic acid It will be particularly noted in connection with the p-cymene that the oxidation product obtained by microbiological means is p-isopropyl benzoic acid. Under circumstances of purely chemical oxidation, this product would not be obtained. Rather, the isopropyl group would become oxidized to the exclusion of the methyl group.

Oxidative microorganisms which may be employed in the practice of the invention include the Nocardia, Pseudomonas, and Mycobacteria. Preferably, we employ the Nocardia.

The substrate employed in the fermentation reaction mixture, in addition to the alkyl substituted cyclic hydrocarbon, is any substrate capable of supporting growth of the particular oxidative microorganism used. The substrate may also be a source of energy, although energy for the microorganisms is supplied by the alkyl substituent on the cyclic hydrocarbon. Suitable substrates include carbohydrates such as starches and sugars. However, sugar-containing mixtures such as molasses may also be employed. Cane molasses or sugar beet molasses may be employed. Corn steep liquor may also be employed.

It is preferred to employ a hydrocarbon as the substrate. We have found that various alkane hydrocarbons provide satisfactory growth of the oxidative microorganisms. The alkane may be a straight-chain alkane or a branched-chain alkane. Included among the alkanes which my be employed are ethane, the isomeric propanes, butanes, pentanes, hexanes, heptanes, octanes, nonanes, decanes, undecanes, dodecanes, tridecanes, tetradecanes, pentadecanes, hexadecanes, heptadecanes, octadecanes, nonadecanes, eicosanes, etc. Preferably, those alkanes which are in the liquid phase are employed.

The fermentation reaction mixture should also contain, in accordance with conventional practice in carrying out microbiological reactions, mineral salts for the growth of the oxidative microorganism. These salts should furnish ammonium or nitrate, potassium, ferrous or ferric, calcium, magnesium, phosphate and sulfate ions, as well as ions of trace elements such as zinc, manganese, copper, and molybdenum. Usually, these mineral salts will be present in sutficient quantities in sugar-containing mixtures such as molasses and corn steep liquor. As indicated subsequently, water is included in the fermentation reaction mixture and most of these mineral salts will usually be present in sufficient quantity in ordinary potable water supplies. Thus, where sugar-containing mixtures, such as molasses or corn steep liquor, or ordinary potable water are employed, separate operations for the provision of these mineral salts may be eliminated. However, it is desirable to add these salts to the fermentation reaction mixture to insure their presence.

The fermentation reaction mixture, during the oxidative reaction, is maintained under conditions to insure optimum growth of the fermentation microorganism. The temperature, for example, should be maintained between about 20 and about 55 C. Preferably, the temperature should be maintained in the neighborhood of 30 C. Further, the pH of the reaction mixture should be maintained near neutrality, namely, at about 7.0. However, the fermentation reaction may be carried out at a pH between about 5.5 and 8.5.

The fermentation reaction mixture will consist primarily of water. The water may constitute 99%, or more, by weight of the liquid phase of the fermentation reaction mixture. However, the water may constitute a much lesser portion of the fermentation reaction mixture. For example, the fermentation reaction mixture may contain as little as 50% by weight of water. Generally, any proportion of water heretofore employed in microbial oxidation of hydrocarbons may be used.

The alkyl substituted cyclic hydrocarbons subjected to the microbial oxidation reaction, as well as the substrate where a hydrocarbon substrate is employed, are insoluble in water. Accordingly, the hydrocarbons will constitute a separate, discrete, phase within the fermentation reaction mixture. In order to accelerate the fermentation reaction, it is desirable to renew continuously the surfaces of the hydrocarbon phase to make the hydrocarbon readily available to the microorganism. Accordingly, it is desirable to maintain the fermentation reaction mixture in a condition of agitation during the oxidative reaction. This may be effected by propellers, paddles, rockers, stirrers, or other means ordinarily employed for effecting agitation of a liquid mixture.

The microbial oxidation reaction requires that oxygen be supplied to the fermentation reaction mixture. Oxygen can be supplied to the fermentation reaction mixture by employing reactors open to the atmosphere. With agitation of the reaction mixture, the surface thereof exposed to the atmosphere is continuously being renewed and oxygen is thereby taken up by the mixture. If desired, oxygen may be supplied by bubbling oxygen, or an oxygen-containing gas such as air, through the fermentation reaction mixture. Further, if desired, for the purpose of avoiding excessive evaporation of water from the fermentation reaction mixture, the oxygen or oxygen-containing gas may be humidified prior to bubbling through the fermentation reaction mixture. It will be realized that, where oxygen or oxygen-containing gas is bubbled through the reaction mixture, the bubbling of the gas may provide the desired agitation of the reaction mixture. 0n the other hand, agitation by other means may additionally be employed in order to insure that the agitation is adequate.

Following the fermentation reaction, the oxidation products of the alkyl substituted hydrocarbon are removed by conventional methods from the fermentation reaction mixture. The fermentation reaction mixture may be subjected to such procedures as may be required to remove the body cells of the microorganism. Suitable procedures include decantation, centrifuging, and filtration. The mixture may also be subjected to such procedures as may be required to remove any remaining hydrocarbon. Extraction of the reaction mixture with a solvent, such as petroleum ether, in which the hydrocarbon, but not the oxidation product, is soluble may be employed. Thereafter the reaction mixture may be extracted with a solvent in which the oxidation products are soluble. With water contained in the reaction mixture, this solvent will be a water-immiscible solvent. Satisfactory results have been obtained by extracting the fermentation reaction mixture with diethylether. The oxidation product may then be recovered from the diethylether or other solvent employed by evaporation or other suitable procedure. The oxidation product may also be recovered from the reaction mixture by steam distillation.

The examples following will be further illustrative of the procedure of the invention.

Example I This example will be illustrative of the oxidation by Nocardia of n-butylcyclohexane to produce cyclohexaneacetic acid. The substrate employed for supporting the growth of the Nocardia was n-octadecane.

To a fermentor were added two liters of aqueous medium containing mineral salts in the following amounts.

Grams per liter Salt of water (NH 50 1.0 KH PO 0.25 Na HPO 0.25 N'a CO 0.1

CaCl 0.01 'FeSO .7H O 0.005 MnSO 0.002

To this aqueous medium were added eight milligrams per liter of ashed yeast extract to insure the presence in the medium of trace elements required for bacterial growth. To the resulting mixture were then added 1.6 grams of n-octadecane. There were also added to the mixture 100 milliliters of a culture of Nocardia. This culture had previously been grown for 48 hours on n-octadecane and on a dry weight basis mounted to 100 milligrams of Nocardia cells.

The mixture was incubated in a water bath at 30 C. for 24 hours. At the end of this time, 0.8 gram of nbutylcyclohexane was added to the mixture. The reaction mixture was then incubated at 30 C. for an additional 148 hours.

During the entire incubation period, the reaction mixture was agitated employing a three-bladed impeller. Also, the fermentor was open to the atmosphere. For 124 hours following the addition to the n-butylcyclohexane, the pH of the reaction mixture was maintained at about 7 by addition thereto of sodium and potassium phosphates.

Following the incubation period, the bacterial cells were removed from the reaction mixture by centrifugation. The total dry weight of these cells was 0.89 gram. The supernatant culture liquor was then passed through a Seitz filter to insure clarity. Dilute sulfuric acid was added to make the liquor acid and the acidified liquor was then steam distilled to remove the cyclohexaneacetic acid. An amount of 0.294 gram of this acid Was recovered. Based on the weight of the n-butylcyclohexane employed, the yield of the cyclohexaneacetic acid equalled 36.8 percent. A greater yield would have been obtained except for loss of n-butylcyclohexane to the atmosphere as a result of the fermentor being open during the fermentation reaction. The same is true in connection with the subsequent examples where loss of the alkyl substituted cyclic hydrocarbon to the atmosphere from the open fermentor occurred during the fermentation reaction.

Example 2 This example will be illustrative of the oxidation by Nocardia of n-dodecylbenzene to produce phenylacetic acid employing n-octadecane as the substrate for supporting the growth of the bacteria.

To a fermentor were added 100 milliliters of the same aqueous medium containing mineral salts described in the previous example. To this aqueous medium were added four milligrams of ashed yeast extract. To the resulting mixture were added 44 milligrams of n-dodecylbenzene and 44 milligrams of n-octadecane. The mixture was then inoculatedwith five milligrams on a dry weight basis of a culture of Nocardia. This culture had previously been grown on n-octadecane. The fermentor was then incubated at 30 C. for 96 hours. During the in cubation period, the fermentor was continuously agitated with a mechanical shaker and was open to the atmosphere.

Following the incubation period, the reaction mixture was subjected to centrifugation to remove the bacterial cells. About 16.3 milligrams of bacterial cells were re Example 3 In this example, n-dodecylbenzene was oxidized to phenylacetic acid employing Nocardia. N-decane was employed as the substrate for growth of the Nocardia.

To 100 milliliters of the same aqueous medium containing mineral salts as described in the preceding examples were added four milligrams of ashed yeast extract. Forty-four milligrams of n-dodecylbenzene were added to the resulting mixture along with the same amount of ndecane. The mixture was inoculated with live milligrams, on a dry weight basis, of a culture of Nocardia which had been previously grown on n-decane. The procedure thereafter was the same as described above in connection with Example 2 and the yield of phenylacetic acid was 6.5 milligrams.

Example 4 In this example, ethylbenzene was oxidized to phenylacetic acid employing Nocardia and n-hexadecane was employed as the substrate for growth of the bacteria.

The procedure followed was the same as described above in connection with Example 2. However, the amount of ethylbenzene and the amount of n-hexadecane were milligrams each. Further, the culture of Nocardia had previously been grown on n-hexadecane. The yield of phenylacetic acid was 8.4 milligrams.

Example 5 In this example, n-butylbenzene was oxidized to phenylacetic acid. Nocardia was the bacteria employed and the substrate for growth of the baccria was n-hexadecane.

The procedure followed was the same as described above in connection with Example 2. The amount of alkyl substituted cyclic hydrocarbon and alkane hydrocarbon substrate was the same as in Example 4. The yield of phenylacetic acid was 10 milligrams.

Example 6 In this example, p-cymene was oxidized to p-isopropylbenzoic acid employing Nocardia and emp oying n-hexadecane as the substrate for growth of the Nocardia.

Two liters of the aqueous medium containing mineral salts to which ashed yeast extract was added as described above in connection with Example 1 were placed in a fermentor. To the mixture was added, on a dry weight basis, five grams of an inoculum of Nocardia previously grown on n-hexadecane. Thereafter, two milliliters of p-cymene and eight grams of n-hexadecane were added to the mixture.

The reaction mixture was incubated at 30 C. for a period of five days during which it was continuously agitated and was open to the atmosphere. Further, during this period, air to which p-cymene vapor was added was bubbled continuously through the mixture. Seventy-two hours after incubation, an additional five milliliters of pcymene were added to the reaction mixture.

Following the termination of incubation period, the fermentation reaction mixture was treated in accordance with the same procedure described above for the fermentation reaction mixture of Example 2. The yield of pisopropylbenzoic acid was 0.52 gram.

Having thus described our invention, it will be understood that such description has been given by way of illustra-tion and example and not by way of limitation, reference for the latter purpose being had to the appended claims.

We claim:

1. A process for the microbiological oxidation of a cyclic hydrocarbon containing an alkyl substituent comprising forming a cyclic carboxylic acid from said hydrocarbon by subjecting said cyclic hydrocarbon to the action of a microorganism that is a hydrocarbon oxidizer and that is selected from the genera consisting of Nocardia, Pseudornonas and Mycobacteria in a fermentation reaction mixture containing in addition to the said cyclic hydrocarbon a substrate capable of supporting the growth of said oxidative microorganism; and recovering the said cyclic carboxylic acid.

2. The process of claim 1 wherein said substrate comprises a carbohydrate.

3. The process of claim 1 wherein the said substrate comprises an alkane hydrocarbon.

4. A process for the microbiological oxidation of a cyclic hydrocarbon containing an alkyl substituent and being selected from the class consisting of toluene, xylene, mesitylene, ethylbenzene, diethylbenzene, propylbenzene, butylbenzene, amylbenzene, hexylbenzene, heptylbenzene, octylbenzene, p-cymene, methylnaphthalene, ethylnaphthalene, methylcyclopentane, dimethylcyclopentane, trimethylcyclopentane, ethylcyclopentane, diethylcyclopentane, propylcyclopentane, butylcyclopentane, amylcyclopentane, hexylcyclopentane, heptylcyclopentane, octylcyclopentane, methylcyclohexane, dimethylcyclohexane, trimethylcyclohexane, tetramethylcyclohexane, ethylcyclohexane, propylcyclohexane, isopropyl-4-methylcyclohexane, butylcyclohexane, amylcyclohexane, hexylcyclohexane, heptylcyclohexane, and octylcyclohexane which process comprises forming a cyclic carboxylic acid from said hydrocarbon by subjecting said cyclic hydrocarbon to the action of a microorganism that is a hydrocarbon oxidizer and that is selected from the genera consisting of Nocardia, Pseudomonas, and Mycobacteria in a fermentation reaction mixture containing in addition to the said cyclic hydrocarbon a substrate capable of supporting the growth of said oxidative microorganism whereby said oxidative microorganism utilizes said alkyl substituent of said cyclic hydrocarbon as a source of energy and effects oxidation of said alkyl substituent as the predominant oxidative reaction and said oxidative microorganism utilizes said substrate for growth; and recovering the said cyclic carboxylic acid.

5. The process of claim 4 wherein said substrate comprises a carbohydrate.

6. The process of claim 4 wherein said substrate comprises an alkane hydrocarbon.

7. The process of claim 4 wherein said substrate comprises an alkane hydrocarbon selected from the class con sisting of ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and eicosane.

8. The process of claim 4 wherein said fermentation reaction mixture is maintained at a temperature between about 20 C. and about C. and a pH between about 5.5 and 8.5 and an oxygen-containing gas is supplied to said fermentation reaction mixture.

References Cited in the file of this patent ticularly relied upon. Published 1954 by Elsevier Press Inc., Houston. 

1. A PROCESS FOR THE MICROBIOLOGICAL OXIDATION OF A CYCLIC HYDROCARBON CONTAINING AN ALKYL SUBSTITUENT COMPRISING FORMING A CYCLIC CARBOXYLIC ACID FROM SAID HYDROCARBON BY SUBJECTING SAID CYCLIC HYDROCARBON TO THE ACTION OF A MICROORGANISM THAT IS A HYDROCABON OXIDZER AND THAT IS SELECTED FROM THE GENERA CONSISTING OF NOCARDIA, PSUEDOMONAS AND MYCOBACTERIA IN A FERMENTATION REACTION MIXTURE CONTAINING IN ADDITION TO THE SAID CYCLIC HYDROCARBON A SUBSTRATE CAPABLE OF SUPPORTING THE GROWTH OF SAID OXIDATIVE MICROORGANISM; RECOVERING THE SAID CYLIC CARBOXYLIC ACID. 